INFECTION AND IMMUNITY, Sept. 2008, p. 3924–3931 Vol. 76, No. 90019-9567/08/$08.00�0 doi:10.1128/IAI.00372-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Alterations of Splenic Architecture in Malaria Are InducedIndependently of Toll-Like Receptors 2, 4, and 9 or

MyD88 and May Affect Antibody Affinity�†Emma T. Cadman,1‡ Asmahan Y. Abdallah,1 Cecile Voisine,1 Anne-Marit Sponaas,1 Patrick Corran,2

Tracey Lamb,1 Douglas Brown,1,3 Francis Ndungu,1 and Jean Langhorne1*Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA,

United Kingdom1; National Institute of Biological Standards and Control, Blanche Lane, South Mimms,Potters Bar, Herts EN6 3QG, United Kingdom2; and Division of Molecular Immunology, National Institute for

Medical Research, The Ridegway, London NW7 1AA, United Kingdom3

Received 24 March 2008/Returned for modification 3 May 2008/Accepted 5 June 2008

Splenic microarchitecture is substantially altered during acute malaria infections, which may affect thedevelopment and regulation of immune responses. Here we investigated whether engagement of host Toll-likereceptor 2 (TLR2), TLR4, TLR9, and the adaptor protein MyD88 is required for induction of the changes andwhether antibody responses are modified when immunization takes place during the period of splenic disrup-tion. The alterations in splenic microarchitecture were maximal shortly after the peak of parasitemia and werenot dependent on engagement of TLR2, TLR4, or TLR9, and they were only minimally affected by the absenceof the MyD88 adaptor molecule. Although germinal centers were formed in infected mice, they did not containthe usual light and dark zones. Immunization of mice with chicken gamma globulin 2 weeks prior to acutePlasmodium chabaudi infection did not affect the quantity or avidity of the immunoglobulin G antibodyresponse to this antigen. However, immunization at the same time as the primary P. chabaudi infection resultedin a clear transient reduction in antibody avidity in the month following immunization. These data suggest thatthe alterations in splenic structure, particularly the germinal centers, may affect the quality of an antibodyresponse during a malaria infection and could impact the development of immunity to malaria or to otherinfections or immunizations given during a malaria infection.

The spleen has a central role in the immune response tomalaria in humans (46) and rodents (20, 30, 67), and spleno-megaly is one of the most striking features of malaria infection.Within the first week after infection the size of the spleenincreases severalfold, due in part to an influx of lymphocytes inboth human (14, 35, 46) and mouse (54, 68) infections. Addi-tionally, malaria-associated splenomegaly has been associatedwith increased erythropoiesis in mice (41, 52, 61, 68). Malarialsplenomegaly is accompanied by transient alterations in themicroarchitecture during acute infection (2, 9, 29, 32, 54, 65,67), which in mouse infections returns to normal several weeksafter the acute parasitemia (1, 54, 67).

The alterations of the splenic microarchitecture observedduring acute malaria infection are similar in some respects tothose observed after administration of the Toll-like receptor 4(TLR4) ligand lipopolysaccharide (LPS) (19). MyD88, an in-tracellular adaptor protein for TLR4 and other TLRs (for areview, see reference 55), as well as other pattern recognitionor cytokine receptors (26), is involved in the initial host re-

sponse to erythrocytic stages of Plasmodium (4, 28, 45). Inaddition, Plasmodium contains ligands that bind to at leastthree TLRs, including glycosylphosphatidylinositol anchors,which have been shown to bind TLR2 and to a lesser extentTLR4 (28), and DNA trapped within hemozoin, which canstimulate dendritic cells via TLR9 (13, 45). Therefore, it ispossible that ligation of TLRs is involved in the changes in thespleen structure in malaria infections.

Changes in the location of plasma cells, B cells, T cells, anddendritic cells could affect access to antigen, interactions be-tween these cells, and access to the chemokines necessary forcorrect migration of cells and thus could impede immune re-sponses. In this regard, there is a wealth of information sug-gesting that immune responses are suppressed in malaria in-fections (10, 16, 37, 42, 48, 64, 69–71) and that both splenic Tand B cells are lost from the spleen and undergo significantapoptosis (2, 22, 53, 71).

Here we investigated the role of TLRs and the adaptorprotein MyD88 in the transient alterations in splenic microar-chitecture that take place during Plasmodium chabaudi infec-tion of mice and observed that splenomegaly and changesin spleen structure occurred independently of TLR2, TLR4,TLR9, or the MyD88 adaptor molecule. The significantchanges in the spleen during the acute infection, including thelack of formation of dark and light zones within the germinalcenters, did not appear to affect the magnitude of an immu-noglobulin G (IgG) antibody response to an unrelated antigen(chicken gamma globulin [CGG]) administered 2 weeks beforea primary infection, but affinity maturation of the anti-CGG

* Corresponding author. Mailing address: Division of Parasitology,National Institute for Medical Research, The Ridgeway, London NW71AA, United Kingdom. Phone: 44 208 816 2558. Fax: 44 208 816 2638.E-mail: jlangho@nimr.mrc.ac.uk.

† Supplemental material for this article may be found at http://iai.asm.org/.

‡ Present address: Royal Veterinary College, Department of Veter-inary Basic Sciences, Royal College Street, London NW1 0TU2,United Kingdom.

� Published ahead of print on 16 June 2008.

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antibody response was delayed in mice that received CGG andP. chabaudi concurrently.

MATERIALS AND METHODS

Mice. Female C57BL/6 mice and TLR2�/�, TLR4�/�, TLR6�/�, TLR9�/�,and MyD88�/� mice (5, 23, 24, 57, 58) with a C57BL/6 background were bred inthe specific-pathogen-free unit of the National Institute for Medical Research(NIMR) under the NIMR guidelines for animal husbandry. They were infectedor immunized when they were 6 to 12 weeks old. For experimental use, all micewere conventionally housed with sterile bedding, food, and water. All experi-ments with mice were carried out under a United Kingdom Home Office licenseaccording to United Kingdom law and were approved by the NIMR EthicalReview Panel.

Parasites and immunization. P. chabaudi chabaudi AS (66) was maintained asa frozen stock and was passaged in mice as described previously (36). Forexperiments, mice were inoculated with 105 parasitized red blood cells (pRBC)diluted in 100 �l Kreb’s saline containing glucose (11 mM) intraperitoneally(i.p.). Infections were monitored by microscopic examination of Giemsa-stainedthin blood films as described previously (31).

To obtain irradiated parasites, mice were exsanguinated at a parasitemia ofapproximately 30% pRBC. Infected blood, diluted 1:10 in Kreb’s saline, wasirradiated (30,000 rads) and resuspended in Kreb’s saline at a concentration ofapproximately 5 � 108 pRBC/ml. Mice were inoculated with three 200-�l dosesof the pRBC suspension given 2 days apart. Spleens were removed for histolog-ical examination 3 days after the final dose. To determine that irradiation hadprevented parasite replication, Giemsa-stained thin blood films from the inocu-lated mice were assessed for the subsequent 10 days for the presence of pRBCand were found to be negative.

Groups of mice (six mice per group) were immunized i.p. once with 25 �g ofCGG in alum. One group received CCG in alum and was not infected, one groupwas given CGG at the time of infection with 105 pRBC, and one group was givenCGG in alum 2 weeks prior to initiation of the P. chabaudi infection. Blood usedto obtain plasma was taken 1, 2, and 3 months after immunization.

Mice were given three 2-�g doses of LPS (Alexis) i.p. in 100 �l phosphate-buffered saline (PBS) on days 0, 2, and 5, and spleens were removed for histo-logical examination (see below) on day 7.

ELISAs for measurement of antibodies to CGG. Enzyme-linked immunosor-bent assays (ELISAs) were carried out essentially as described previously (3),except that 5 �g/ml CGG in 0.2 M sodium bicarbonate (pH 9.6) was used as thecoating antigen and alkaline phosphatase-conjugated goat anti-mouse IgG(Southern Biotechnology) (with p-nitrophenyl phosphate [Sigma] as the sub-strate) was used as the detecting reagent. Optical densities at 450 nm weredetermined. A pooled plasma sample taken from mice 2 weeks after the thirdimmunization with 25 �g of CGG was used as a standard, and normal mouseplasma was used as the negative control. Antibody amounts were expressed inarbitrary units of specific antibody relative to the standard (1,000 U).

Measurement of antibody avidity. The avidity of CGG-specific antibody wasmeasured by determining the effect on the formation of an antibody-antigencomplex of 1 M ammonium thiocyanate (NH4SCN). CGG at a concentration of5 �g/ml diluted in 15 mM Na2CO3–34 mM NaHCO3 (pH 9.4) was used as acoating antigen. ELISAs were carried out as described above using twofolddilutions of plasma from CGG-immunized mice, starting with a dilution prede-termined to give maximum binding to the plate-bound antigen, in the absence ofNH4SCN; the test series was diluted in 1 M NH4SCN in PBS, and a control serieswas diluted in 1% bovine serum albumin-PBS-Tween 20. After washing, boundantibody was detected with an anti-mouse IgG-horseradish peroxidase conjugate(Dako) and the o-phenylenediamine dihydrochloride (Sigma) substrate. Opticaldensities at 490 nm were determined. The titration curves were plots of absorp-tion (A) versus log reciprocal dilution (D). Binding curves were fitted to theequation A � Amax · [1 � D/(D � Ke)] � B, where A is the observed absorptionat dilution 1/D, Amax is the absorption when CGG is saturated with antibody, Bis the blank value, and Ke is the midpoint titer of the serum [the estimateddilution giving 50% binding corresponding to the inflection point in the plot ofA versus log(D)]. Fitting was carried out by least-squares minimization using thesolver attachment in Excel, and the goodness of fit was tested by regressionanalysis (r2 � 0.7 was considered acceptable). The titration curve was shifted tothe left by increasing concentrations of NH4SCN (47), and the avidity of theanti-CGG antibody response was defined as the ratio of the fitted K estimate(KC) in the presence of 1 M NH4SCN to the K estimate in the absence ofchaotrope (K0).

Flow cytometry. Spleen cells from naıve or P. chabaudi-infected mice weremashed through a 0.7-�m sieve (Falcon) to create a single-celled suspension.

Erythrocytes were lysed with red blood cell lysis buffer (Sigma), and the cellpellet was washed and resuspended in FACS buffer (1% [wt/vol] bovine serumalbumin, 5 mM EDTA, and 0.01% sodium azide in PBS). Cells were enumeratedwith a hemocytometer and trypan blue (Sigma) exclusion or with a CoulterCounter. Before addition of specific fluorescently labeled antibodies, 5 � 105

cells were preincubated at room temperature for 10 min with anti-Fc receptorantibody (63) to prevent nonspecific binding via the FcR. All other preparationswith antibodies were incubated for 20 min on ice. Cells were analyzed with a BDFACScalibur.

Histology. Five-micrometer spleen sections were prepared as described previ-ously (2). Immunofluorescence staining was carried out by using a methodmodified from the method described by Achtman et al. (2). All antibody incu-bations were carried out in the dark for 1 h. The primary antibodies were ratanti-mouse CD8, CD138, biotinylated hamster anti-mouse CD11c (BD), ratanti-mouse CD3, IgM, CD169/sialoadhesin/MOMA-1, MAdCAM-1 (Serotec),hamster anti-mouse CD11c Alexa Fluor 488 (Caltag), sheep anti-mouse IgD(The Binding Site), biotinylated peanut agglutinin (Vector Labs), rat anti-mouseER-TR9 (BMA), rat anti-mouse F4/80 fluorescein isothiocyanate (FITC)(eBioscience), and goat anti-mouse IgM FITC (Sigma). The secondary antibod-ies used were donkey anti-sheep Alexa Fluor 647, donkey anti-sheep Alexa Fluor568, goat anti-rat IgG Alexa Fluor 488, chicken anti-rat Alexa Fluor 647, Neu-travidin Texas Red (Molecular Probes), donkey anti-sheep horseradish peroxi-dase (The Binding Site), biotinylated rabbit anti-rat IgG (Dako), and streptavi-din FITC (BD). Slides were mounted with fluorescence mounting medium(Dako). Nonfluorescent histological examination was carried out as describedpreviously (2).

Statistics. Differences in splenocyte and follicle numbers between wild-type,TLR�/�, and MyD88�/� mice and differences in antibody avidities (K0 � K1M)were analyzed by using the Mann-Whitney test. Parasitemias for strains of micewere compared by using a one-way analysis of variance. Avidity binding curvefitting was carried out by least-squares minimization, and the goodness of fit wastested by regression analysis.

RESULTS

Alterations in splenic structure during acute malaria infec-tion. The splenic microarchitecture was investigated during thefirst 10 days of P. chabaudi infection, up to and including thepeak of parasitemia (see Fig. S1A in the supplemental mate-rial). As described previously (2), P. chabaudi infection in-duced an increase in spleen size and cellularity (see Fig. S1B inthe supplemental material), as well as a transient loss of clearlydefined follicles including CD3� T-cell and IgD� B-cell areasand transient increases in the number of CD138� plasma cellsand the size of PNA� germinal centers (see Fig. S1C and D inthe supplemental material).

Changes in spleen structure were first visible at day 5 postin-fection, and the maximum alterations were observed on day 10,2 to 3 days after the peak of infection. The clear marginal zonessurrounding the follicles seen in normal spleens disappeared,and there was loss of CD169� marginal metallophilic macro-phages (MMM) and ER-TR9� marginal zone macrophages(MZM) (Fig. 1A). Expression of the adhesion molecule MAd-CAM-1, normally seen as a defined ring surrounding the fol-licles and white pulp (27), was increased and appeared asdiffusely stained areas over the white pulp (Fig. 1A). There wasalso a large increase in the number of F4/80� macrophages,which remained in the red pulp throughout infection (Fig. 1B).Normal splenic structure returned by day 60, when the para-sitemia subsided to subpatent levels (2, 3, 8, 29, 54, 67; data notshown).

The majority of the alterations in splenic microarchitecturewere the result of a live infection, as administration of threedoses of irradiated parasites at 2-day intervals (a total of 3 �108 parasites) to mice did not disrupt the segregation of B- andT-cell zones or result in loss of marginal zone cells (Fig. 1C).

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FIG. 1. Changes in splenic microarchitechure and composition during the acute stage of a primary infection with P. chabaudi and after injectionof irradiated parasites. Five-micrometer-thick cryostat sections of spleens from naıve mice and from mice on day 10 postinfection were stained withIgD (red or brown) and either (A) CD169 (also known as sialoadhesin or MOMA-1) (green), ER-TR9 (blue), or MAdCAM-1 (green) or with (B,upper panel) IgD (red) and F4/80 (green). (B, lower panel) Graph showing quantification of the F4/80 cells enumerated by flow cytometry duringthe first 20 days of infection. The bars indicate the medians and ranges for three mice. (C) Mice were inoculated with irradiated parasites asdescribed in Materials and Methods. Spleens were removed 3 days after the final injection, sectioned, and stained for IgD (red) and for CD3(green), CD169 (green), CD138 (green), peanut agglutinin (green), or MAdCAM-1 (green). The sections in panels A to C are representative ofthree mice. Final magnification, �120.

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However, compatible with exposure to an immunogen(s),there was development of both plasma cells and germinal cen-ters, as well as increased expression of MAdCAM-1 over thewhite pulp.

TLR2, TLR4, TLR6, or TLR9 or the adaptor protein MyD88is not necessary for alterations of the splenic microarchitec-ture during infection. TLR2�/�, TLR4�/�, TLR9�/�, andMyD88�/� mice were infected with P. chabaudi, and theirspleens were subjected to a histological examination at day 10postinfection, when alterations of the splenic microarchitec-ture were maximal. Spleens of uninfected wild-type mice andMyD88�/� and TLR�/� mice contained comparable numberof cells (Fig. 2A) and had similar architecture (see Fig. S2 inthe supplemental material), although the TLR2�/� mousespleens contained significantly fewer cells at day 10 postinfec-tion than the spleens of wild-type mice (P � 0.0286, Mann-Whitney test).

The increase in spleen size or cellularity after infection withP. chabaudi was not dependent on the presence of MyD88,TLR4, or TLR9 despite the slightly higher parasitemia at day10 in infected MyD88�/� mice compared with the other groupsof TLR�/� mice (for MyD88�/� and TLR2�/� mice, P � 0.01;for MyD88 and TLR9�/� mice, P � 0.05 [one-way analysis ofvariance]) (Fig. 2B).

The loss of definition of B- and T-cell areas in the TLR�/�

mice was comparable to that in wild-type C57BL/6 mice. Sim-ilarly, marginal zone structure was disturbed, as determined byloss of sialoadhesin-positive MMM (sialoadhesin is equivalent

to CD169/MOMA-1 shown in Fig. 1A). The changes in MAd-CAM-1 expression and production of CD138� plasma cells inthe TLR�/� and MyD88�/� mice were also similar in all casesto the changes observed in infected C57BL/6 mice (Fig. 3),suggesting that ligation of the individual TLRs by parasitemoieties was not responsible for the changes in the marginalzones. There appeared to be more discernible follicles (num-ber of follicles per 4,720 �m2) in the spleens of infectedMyD88�/� mice than in the spleens of the infected TLR�/�

and wild-type mice (Fig. 2C). However, the differences werenot significant (e.g., for MyD88�/� mice versus wild-type miceon day 10, P � 0.2286 [Mann-Whitney test]; for MyD88�/�

mice versus TLR2�/� mice, P � 0.1).Administration of multiple doses of the TLR4 ligand LPS

(three 2-�g doses, with the spleens removed 3 days after thefinal dose) caused emigration of T cells from the white pulp tothe red pulp, as described previously (19). MAdCAM-1 expres-sion was induced over the white pulp, but in contrast to malariainfection, there was no loss of MMM from the marginal zone;instead, there was a thickening of the MMM ring in the mar-ginal zone (see Fig. S3 in the supplemental material).

Germinal center formation and IgG antibody responsestake place despite changes in the splenic structure. The loss ofmarginal zones and clear definition of T- and B-cell areas maybe indications of adverse alterations in NF-�B and chemokinesignaling (21, 62), which could affect correct cell migration (39,40) and thus immune responses in acutely infected mice. Ger-minal centers did develop during the primary P. chabaudi in-fection (2) (see Fig. S1 in the supplemental material), butdistinct dark and light zones (detected by antibodies to CD35[6]) within these germinal centers, which are normally seen ingerminal centers after immunization (6, 7), were not observedin spleen sections obtained from mice infected with P.chabaudi at 10 days postinfection (Fig. 4A) (typically, 12 ger-minal centers in a 4,720-�m2 area were counted). It is there-fore possible that IgG antibody responses arising from germi-nal center B cells could be impaired during infection. Despitethe abnormal appearance of the germinal centers in infectedmice, the magnitude of the IgG antibody response to CGGthat was administered before (14 days) or at initiation of theacute infection was not noticeably reduced (Fig. 4B). However,the avidity of the anti-CGG IgG antibody response at 1 monthpostimmunization was significantly lower (greater K0/KC ratio)in mice immunized with CGG at the same time that the P.chabaudi infection was initiated than in mice immunized withCGG 2 weeks prior to infection (P � 0.0079, Mann-Whitneytest). This effect was transient as by 2 and 3 months afterimmunization, the avidities of the CGG antibodies were sim-ilar in all groups (Fig. 4C).

DISCUSSION

An acute blood-stage malaria infection in mice results inprofound changes in splenic architecture (1, 9, 29, 54, 67),which may impact the host’s ability to mount a successfulimmune response to the parasite. In addition to the loss ofmarginal zones (MMM, marginal zone B cells, and MZM) andthe general loss of follicular structure observed here in P.chabaudi infections and reported by other workers (1, 9, 54),there is also a clear change in the pattern of expression of the

FIG. 2. P. chabaudi parasitemia, total spleen cell numbers, andnumbers of discernible splenic follicles at 10 days postinfection.C57BL/6 (WT), TLR2�/�, TLR4�/�, TLR9�/�, and MyD88�/� micewere used in the experiments. (A) Total spleen cell numbers in naıvemice on days 7 and 10 postinfection. (B) Percentage of parasitemia atday 10 postinfection. (C) Number of discernible splenic follicles in4,720 �m2 at day 10 postinfection (d10). The bars and error barsindicate the medians and ranges for three mice. D0, day 0.

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mucosal addressin adhesion molecule MAdCAM-1, particu-larly over the remnants of the follicles. These effects are tran-sient, and the spleen returns to its normal size and structurewithin 6 weeks after the primary infection. However, duringthe early acute infection, as the splenic architecture is chang-ing, affinity maturation of the antibody response to a third-party antigen, CGG, is delayed.

Some of the splenic alterations observed in this P. chabaudiinfection, in particular the loss of B cells from the marginalzone, the redistribution of T cells throughout the red pulp, andthe increase in red pulp macrophages, have also been observedin human Plasmodium falciparum infections (65). Such alter-ations of the splenic microarchitecture are, however, not con-fined to malaria infections. They have been observed to agreater or lesser degree in viral, bacterial, and other protozoaninfections and after injection of LPS, a bacterial ligand forTLR2/4 (11, 17, 19). It is possible, therefore, that these alter-ations are initiated through pattern recognition receptor(PRR) recognition of pathogen-associated molecular patterns(PAMPs) as part of the host response to a systemic pathogento allow the cells of the host’s innate immune system to destroythe pathogen and to activate the appropriate acquired immuneresponse. Plasmodium contains many membrane proteins withglycosylphosphatidylinositol anchors, which are recognized byTLR2 and TLR4 (28), and infected red blood cells containhemozoin with trapped DNA, which is recognized by TLR9

(13, 44, 45). However, the absence of these TLRs or MyD88,one the major adaptor proteins in the signaling pathway ofTLRs (for a review, see reference 56) had no significant effecton either the malaria-induced alterations in splenic microar-chitecture or splenomegaly in P. chabaudi-infected mice. Liga-tion of TLRs can affect cell migration and expression of che-mokines (43, 49); however, only a lack of TLR2 had a minoreffect on splenomegaly in P. chabaudi-infected mice. Our re-sults suggest several possibilities: signaling through other TLRsor several TLRs in concert is required for significant splenicchanges, other PAMP-PRR interactions are involved, or theresponse is independent of PAMP-PRR interactions. This is inline with recent studies of P. chabaudi and Plasmodium bergheiinfections (18, 60; C. Voisine and J. Langhorne, unpublisheddata), which, although they did not examine splenomegaly andspleen structure, showed that there were only minor links or noassociation between MyD88, TLR2, TLR4, TLR6, or TLR9expression and susceptibility to malaria infections. Similarly,they could not rule out the possibility of other PAMP-PRRinteractions.

The changes in the spleen structure, particularly the loss ofcells from the marginal zones, does not necessarily mean thatthe cells are no longer in the spleen or that they are notfunctional. Migration out of the marginal zones could be partof a normal protective host immune response, which is simplyexaggerated in the case of malaria by the sustained high num-

FIG. 3. Alteration in splenic microarchitecture during an acute P. chabaudi infection is not affected by a lack of MyD88, TLR2, TLR4, or TLR9.Five-micrometer cryostat sections of spleens from naıve C57BL/6 (WT Naive) and day 10 postinfection C57BL/6 (WT), TLR2�/�, TLR4�/�,TLR9�/�, and MyD88�/� mice were stained with IgD (red) and (A) CD3 (green), (B) sialoadhesin (also known as CD169 or MOMA-1) (green),(C) CD138 (green), or (D) MAdCAM-1 (green). Final magnification, �120. Representative areas for three mice are shown.

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bers of parasites, red cell loss, and increased hematopoesis.Marginal zone B cells, although no longer present in the mar-ginal zones in acute P. chabaudi infection, are still present inthe spleen (2) and thus could still participate in antiparasiteresponses. The location of MMM and MZM in the marginalsinus is ideal for allowing them to move into the red pulp tophagocytose blood-borne antigens, such as pRBC, removingthem from the circulation. From other immunization studiesthese cells are known to be important contributors to the earlyantibody response to blood-borne antigens (for a review, seereference 34). Although MMM have been shown to colocalizewith apoptotic bodies (9), indicating that they are lost from thespleen, it is not clear whether this is before or after phagocy-tosis of pRBC.

The increase in MAdCAM-1 is interesting. Expression ofMAdCAM1, a ligand for L-selectin present on most leukocytes(for a review, see reference 59), is controlled by the alternativeNF-�B pathway (21) and is upregulated in response to inflam-mation and agents such as LPS, tumor necrosis factor alpha,

and interleukin-1 (50). Although MAdCAM-1 plays an impor-tant role in cell trafficking in the gut, no such role has ever beendemonstrated for this molecule in the spleen, and the functionthat it has in the marginal sinus remains unknown (27). Highlevels of expression of this addressin are constitutively foundon high endothelial venules (HEV) in Peyer’s patches andmesenteric lymph nodes, but this molecule is otherwise nor-mally only seen during the early development of HEV (25).Although fenestrated capillaries rather than HEV are presentin the spleen, it is possible that elevated and extensive expres-sion in an infected spleen reflects restructuring of capillariesand compartmentalization of distinct areas to maximize thechances of appropriate host responses and protection of thehematopoetic beds (67) in the red pulp (52).

On the other hand, the alterations of the splenic microar-chitecture seen during acute malaria infection resemble thoseobserved in mice lacking lymphotoxin- expression on B cells(62), which have reduced numbers of B-cell follicles, littledistinction between B- and T-cell zones, and few MMM,MZM, and marginal zone B cells in the marginal zone, as wellas increased MAdCAM-1 expression over the white pulp area.A lack of lymphotoxin- has been shown to increase suscepti-bility to Leishmania and viral infections, secondary to alter-ations in splenic microarchitecture (51), suggesting that thealterations seen in acute malaria infections may be detrimentalto the host and may contribute to the lower B- and T-cellresponses reported in several experimental malaria infectionsand in human infections (15, 38, 42, 64, 71).

Very little is known about whether the modified splenicstructure in malaria infections affects immune responses andhematopoeisis. Although there was a substantial loss of clearlydefined T- and B-cell areas, germinal centers were formed andpersisted for up to 60 days postinfection (1), indicating that aT-cell-dependent B-cell response was able to take place. In-deed, the IgG antibody response to another antigen (CGG)given to mice either at the time of P. chabaudi infection or 2weeks prior to infection was similar in magnitude to that of theantibody response to CGG given in the absence of infection.This contrasts somewhat with the results of Millington et al.(37), who showed that mice immunized with ovalbumin andLPS 6 h or 12 days after malaria infection had significantlyreduced IgG responses to ovalbumin at 3 weeks postimmuni-zation. This may have been due to differences in the timing ofimmunization and the adjuvant used, since in the study of theseworkers, when mice were immunized 4 days after malaria in-fection, no significant differences in IgG responses were ob-served. Thus, whatever effect malaria infection has on gener-ation of antibody responses, the time of immunization matters.This may be a reflection of when splenic changes are initiatedand their transient nature.

Although antibody titers did not appear to be affected bymalaria infection, there was a significant delay in affinity mat-uration when infection and immunization were carried outconcurrently. Normal dark and light zones were not observedwithin the germinal centers in spleens of P. chabaudi-infectedmice, similar to the results for germinal centers in P. berghei-infected mice (12). The light zones of germinal centers containthe network of follicular dendritic cells. Interaction of theimmunoglobulin receptor of B cells with antigen displayed ontheir surface promotes somatic hypermutation of the receptor

FIG. 4. Avidity, but not magnitude, of the antibody response toCGG is reduced by infection with P. chabaudi. (A) Dark and lightzones of germinal center in a P. chabaudi-infected mouse at day 10postinfection. Five-micrometer cryostat sections of spleens from threemice were stained with IgD (red) and CD35 (green). (B and C)Amount (B) and avidity (C) of CGG-specific IgG antibodies of sixmice immunized with CGG in alum (gray bars), mice immunized withCGG in alum given at the same time as primary infection with 105 P.chabaudi parasites (cross-hatched bars), and mice immunized withCGG in alum given 2 weeks prior to initiation of a P. chabaudi infec-tion (open bars). Antibody levels and avidity were determined 1 to 3months postimmunization. Each box and whisker plot in panel Cindicates the range and 95% confidence interval of the values (K0/KCratio, as described in Materials and Methods) obtained for five mice.The horizontal line in each bar indicates the median. Ab, antibody.

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and thus affinity maturation of the antibody response (for areview, see reference 33). A detailed study of follicular den-dritic cells and germinal center reactions together with ananalysis of antibody affinity from individual B cells within ger-minal centers in malaria infections would elucidate whetherthe altered structure of germinal centers plays any role in thedelayed affinity maturation.

The results obtained with the P. chabaudi malaria modelhave important implications in areas where transmission ofmalaria is endemic. Immunization during acute infection, par-ticularly immunization of children, could result in antibodieswith lower affinity. Studies are under way to establish whetherimmunization against a second infection during acute malarialeads to reduced protective efficacy.

ACKNOWLEDGMENTS

This work was supported by the Medical Research Council, UnitedKingdom, and was part of the activities of the BioMalPar EuropeanNetwork of Excellence supported by FP6 EU grant LSHP-CT-2004-503578. Emma T. Cadman received an MRC Ph.D. studentship, andAsmahan Abdallah was a recipient of FP6 EU-funded Marie CurieAction MalPar training (grant MEST-CT-2005-020492).

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